Powdery Mildew Resistance Providing Genes in Cucumis Melo

ABSTRACT

The present invention relates to powdery mildew resistance providing genes of the  Cucumis  family, and especially  Cucumis melo,  wherein said resistance is provided by impairment of the present genes. Further, the present invention relates plants comprising the present impaired resistance conferring genes and seeds, embryos or other propagation material thereof. Especially, the present invention relates to powdery mildew resistance conferring genes, wherein the amino acid sequence encoded by said resistance conferring gene is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID. No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/002,259, filed Aug. 29, 2013, which is the United Statesnational phase of International Application No. PCT/EP2012/053208 filedFeb. 24, 2012, and claims priority to International Application No.PCT/EP2011/053053, filed Mar. 1, 2011, the disclosures of which arehereby incorporated by reference in their entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 1704760_ST25.txt. The size of thetext file is 57,737 bytes, and the text file was created on May 22,2017.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to powdery mildew resistance providinggenes of Cucumis melo, wherein said resistance is provided by impairmentof the present genes either at the expression or protein level. Further,the present invention relates to plants comprising the presentresistance conferring genes and seeds, embryos or other propagationmaterial thereof.

Powdery mildew (PM) is one of the main fungal diseases known in plantsbelonging to the Cucumis family such as Cucumis melo (melon), both inthe field and greenhouse.

Powdery mildew diseases are generally caused by many different speciesof fungi of the order Erysiphales. The disease is characterized bydistinctive symptoms such as white powder-like spots on the leaves andstems. Generally, the lower leaves are the most affected, but the mildewcan appear on any part of the plant that is exposed above ground. As thedisease progresses, the spots get larger and thicker as massive numbersof spores form, and the mildew spreads up and down the length of theplant such on the stem and even the fruits.

Severely affected leaves can become dry and brittle, or can wither anddie. Because of the infection, the fruits can be smaller in size, fewerin number, less able to be successfully stored, sun scalded,incompletely ripe, and having a poor flavor. It may also predisposeplants to be more vulnerable to other pathogens. Eventually, the plantcan die.

Powdery mildew can, amongst others, be caused by the fungus Sphaerothecafuliginea (recently renamed: Podosphaera xanthii also designated asOidium erysiphoides) and/or Erysiphe cichoracearum DC (recently renamed:Golovinomyces cichoracearum also designated as Oidium chrysanthemi).

Considering the economic importance of Cucumis plant species, such asmelon, there is a continued need in the art to provide powdery mildewresistance providing genes.

In view of the above need in the art, it is an object of the presentinvention, amongst other objects, to meet this need.

SUMMARY OF THE INVENTION

According to the present invention, this object, amongst other objects,is met by an powdery mildew resistance conferring gene as defined in theappended claim 1.

Specifically, this object of the present invention, amongst otherobjects, is met by a powdery mildew resistance conferring gene, whereinthe amino acid sequence encoded by said resistance conferring gene isselected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ IDNo. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, andamino acid sequences with more than 70% identity, preferably more than80% identity, more preferably more than 90% identity, and mostpreferably more than 95% identity such as more than 96%, 97%, 98%, 99%;and wherein said resistance conferring gene is impaired.

The object of the present invention, amongst other objects, isadditionally met by a powdery mildew resistance conferring gene, whereinthe cDNA sequence transcribed from said resistance conferring gene isselected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, andcDNA sequences with more than 70% identity, preferably more than 80%identity, more preferably more than 90% identity, and most preferablymore than 95% identity such as more than 96%, 97%, 98%, 99%; and whereinsaid resistance conferring gene is impaired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the results of the complementation assay for35S::CmKIP1_cDNA (designated 35S::CmMlo1_cDNA) which resulted in again-of-function for the powdery mildew susceptibility pathway. Thepercentage of diseased leaf area 12 days post inoculation is shown forArabidopsis WT, Atmlo2/6 and Atmlo2/6/12 mutants, the data representmean±s.d. of 2 individuals. For Atmlo2+35S::CmKIP1_cDNA,Atmlo2/6+35S::CmKIP1_cDNA and Atmlo2/6/12+35S::CmKIP1_cDNA the datarepresent mean±s.d. of respectively 4, 4 and 9 primary transformants;

FIG. 2: shows the results of the complementation assay for35S::CmKIP1_cDNA (designated 35S::CmMlo1_cDNA) which resulted in again-of-function for the powdery mildew susceptibility pathway. Thelevel of resistance observed for Atmlo2/6 and Atmlo2/6/12 mutants andfor primary transformants Atmlo2+35S::35S::CmKIP1_cDNA,Atmlo2/6+35S::35S::CmKIP1_cDNA and Atmlo2/6/12+35S::35S::CmKIP1_cDNA ispresented in comparison to the Arabidopsis WT phenotype;

FIG. 3: shows complementation of Atmlo2/6/12 with the 35S::CmKIP2_cDNAwhich results in gain-of-function for the powdery mildew susceptibilitypathway. (A) Susceptible interaction phenotype of G. orontii on leavesof WT Columbia; clear hyphae and conidiophore development. (B) Resistantinteraction phenotype of G. orontii on leaves of Atmlo2/6/12; spores onthe leaf surface have germinated but no further development is observed.(C, D, E) Intermediate interaction phenotypes observed on Atmlo2/6/12after complementation with 35S::CmKIP2_cDNA; on some primarytransformants the germinated spores do not further develop (C), on otherprimary transformants germination of spores is observed, hyphae dodevelop and conidiophores are formed (D,E).

DESCRIPTION OF THE INVENTION

Impaired resistance conferring gene according to the present inventionis meant to indicate a gene providing a reduced, or even absent,susceptibility to powdery mildew caused by fungi indicated bypowder-like spots on the leaves and stems, such as fungi belonging tothe order Erysiphales such as Sphaerotheca fuliginea (recently renamed:Podosphaera xanthii also designated as Oidium erysiphoides) and/orErysiphe cichoracearum DC.

Impaired resistance conferring gene according to the present inventionare mutated genes. The mutation of the present genes can throughdifferent mechanisms results in impairment. For example, mutations inprotein encoding DNA sequences may lead to mutated, truncated ornon-functional proteins. Mutations in non-coding DNA sequences may causealternative splicing, translation or protein trafficing. Alternatively,a mutation resulting in an altered transcriptional activity of a gene,which determines the amount of mRNA available for translation toprotein, may results in low levels, or absence, of proteins.Additionally, the impairment of gene function may be caused aftertranslation, i.e. at protein level.

Impairment according to the present invention is also indicated byobserving a powdery mildew resistance in a Cucumis melo plant comprisinga gene which as mutated at the protein level as compared to the SEQ IDNos. provided herein or no expression of the SEQ ID Nos. provided hereinis observed.

Impaired is also indicated herein as a non-functional gene or protein.Although the function of the present genes is not yet identified, anon-functional gene or protein can be readily determined by establishingpowdery mildew resitance (non-functional) or powdery mildewsusceptibility (functional) in a plant. A powdery mildew resitance(non-functional) plant is indicated by comprising a gene which asmutated at the protein level as compared to the SEQ ID Nos. providedherein or no expression of the SEQ ID Nos. provided herein is observed.

Functional and non-functional genes or proteins can also be determinedusing complementation experiments. For example, transforming a resistantpowdery mildew Cucumis melo plant with any of the present genes orproteins will result in a powdery mildew susceptible Cucumis melo plantwhen the gene or protein is functional while the Cucumis melo plant willremain resistant when the gene or protein is non-functional.

According to the present invention, the present powdery mildewresistance conferring genes provide powdery mildew resistance when thepresent genes are impaired. Impaired according to the present inventioncan be indicated by the absence, or decrease of a functional, ornon-muted, protein identified herein as SEQ ID No. 2, SEQ ID No. 4, SEQID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14.In the art, many mechanisms are known resulting in the impairment of agene either at the transcription, translation or protein level.

For example, impairment at the transcription level can be the result ofone or more mutations in transcription regulation sequences, such aspromoters, enhancers, and initiation, termination or intron splicingsequences. These sequences are generally located 5′ of, 3′ of, or withinthe coding sequence represented by SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13.Impairment can also be provided by a deletion, rearrangement orinsertion in the present genes.

Impairment at the translation level can be provided by a prematurestop-codons or other RNA→protein controlling mechanisms (such assplicing) or posttranslational modifications influencing, for example,protein folding or cellular trafficking.

Impairment at the protein level can be provided by truncated, misfoldedor disturbed protein-protein interactions.

Independent of the underlying mechanism, impairment according to thepresent invention is indicated by an decrease or absence a functionalprotein according to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ IDNo. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14.

According to a preferred embodiment, impairment according to the presentinvention is provided by one or more mutations in the present genesresulting in the absence of a protein expression product. As indicated,these mutations can cause a defective expression at the transcription ortranslation level.

According to another preferred embodiment, impairment according to thepresent invention is caused by one or more mutations in the presentgenes resulting in a non-functional protein expression product. Anon-functional protein expression product can, for example, be caused bypremature stop-codons, incorrect translation or post-translationalprocessing or by insertions, deletions or amino acid changes.

Using molecular biology methods, impairment of the present genes canalso be accomplished by gene silencing, for example using siRNA orknocking out of the present genes. Methods based on EMS or othermutagenic chemical compounds capable of randomly change nucleic acidsinto other nucleotides are also contemplated within the context of thepresent invention. Detection of such mutations typically involves highsensitivity melting curve analyses or nucleotide sequencing-basedTILLING procedures.

The present invention relates to nucleotide and amino acid sequenceswith more than 70%, preferably more than 80%, more preferably more than90% and most preferably more than 95% sequence identity either at thenucleotide level or the amino acid level.

Sequence identity as used herein is defined as the number of identicalconsecutive aligned nucleotides, or amino acids, over the full length ofthe present sequences divided by the number of nucleotides, or aminoacids, of the full length of the present sequences and multiplied by100%. For example, a sequence with 80% identity to SEQ ID No. 1comprises over the total length of 1713 nucleotides of SEQ ID No. 1 1370or 1371 identical aligned consecutive nucleotides, i.e., 1370 or1371/1713*100%=80%.

According to the invention, the present genes are derived from Cucumismelo.

According to another aspect, the present invention relates to Cucumismelo plants comprising in their genome the present impaired powderymildew resistance conferring genes, i.e., plants not expressing afunctional protein selected from the group consisting of SEQ ID No. 2,SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12and SEQ ID No. 14, and amino acid sequences with more than 70% identity,preferably more than 80% identity, more preferably more than 90%identity, and most preferably more than 95% identity.

In general, and preferably, the present plants will be homozygous forthe present impaired genes, i.e., comprising two impaired powdery mildewresistance conferring genes, wherein the cDNA sequence transcribed fromsaid resistance conferring gene is selected from the group consisting ofSEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9,SEQ ID No. 11 and SEQ ID No. 13, and cDNA sequences with more than 70%identity, preferably more than 80% identity, more preferably more than90% identity, and most preferably more than 95% identity.

Considering the benefits of the present plants, i.e., providing powderymildew resistance in melon plants, the invention also relates to seeds,plant parts or propagation material capable of providing the presentpowdery mildew resistant melon plants which seeds, plant parts orpropagation material comprise one or more of the present powdery mildewresistance conferring genes, i.e., impaired powdery mildew resistanceconferring genes, wherein the cDNA sequence transcribed from saidresistance conferring gene is selected from the group consisting of SEQID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ IDNo. 11 and SEQ ID No. 13, and cDNA sequences with more than 70%identity, preferably more than 80% identity, more preferably more than90% identity, and most preferably more than 95% identity.

According to yet another aspect, the present invention relates toisolated nucleotide sequences selected from the group consisting of SEQID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ IDNo. 11 and SEQ ID No. 13, and nucleotide sequences with more than 70%identity, preferably more than 80% identity, more preferably more than90% identity, and most preferably more than 95% identity.

According to still another aspect, the present invention relates toisolated amino acid sequences selected from the group consisting of SEQID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70%identity, preferably more than 80% identity, more preferably more than90% identity, and most preferably more than 95% identity.

The present invention also relates to the use of one or more of thepresent powdery mildew resistance conferring genes, one or more of thepresent isolated nucleotide sequences, or one or more of the presentisolated amino acid sequences for providing a powdery mildew resistantmelon plants (Cucumis melo). As indicated, the present use is based onimpairment, either at the expression or protein level, of the genesdescribed herein and can be readily determined by the presently providedcDNA and amino acid sequences optionally in combination withdetermination of the presence or absence of powdery mildew resistanceand/or in combination with complementation assays.

The present invention will be further described in the examples below ofpreferred embodiments of the present invention. In the example,reference is made to figures wherein:

EXAMPLES Example 1 Complementation of Arabidopsis Thaliana Atmlo MutantPlants Material and Methods

Full length cDNA of melon CmKIP genes were cloned either in pBINPLUS orGateway vectors under control of a 35S promoter using standard cloningtechniques.

In order to analyze the in planta function of identified melon powderymildew resistance genes, via the Nottingham Arabidopsis Stock Centre(University of Nottingham, Sutton Bonington Campus, Loughborough, LE125RD, United Kingdom), seeds were obtained of Arabidopsis thalianaecotype Columbia (Col) mutant line NACS ID N9707 (mlo2-5 single mutant),N9710 (mlo2-5, mlo6-2 double mutant) and N9715 (mlo2-5, mlo6-2, mlo12-1triple mutant).

The seeds were cultivated in soil, DNA was extracted and PCR assays withprimers flanking the mutations (Table 1) were used to screen 4-week-oldplants for the presence of the mutations as described at The ArabidopsisInformation Resource (TAIR).

TABLE 1Primer pairs used to identify WT gene versus mutated Mlo homologue inArabidopsis mutant lines (5′ to 3′). Primer pair WT genePrimer pair mutated gene Mlo2-5 acgtggaagtcgtggg attcgttaccgggagcttcataaccaatctcgat acgtggaagtcgtggg aggaaga  aaaatgc  acac  aggaaga (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) Mlo6-2ttcataaccaatctcgat acaagaactggtttcat acac  ttagca  (SEQ ID NO: 19)(SEQ ID NO: 20) Mlo12-1 tggagcaagtctacctt tcagtgggctgcattcaggtgcagcaaaaccc tggagcaagtctacctt taccctctgg  cacaaa  acacttttacttctaccctctgg  (SEQ ID NO: 21 (SEQ ID NO: 22) (SEQ ID NO: 23)(SEQ ID NO: 24)

The floral dip method was used to transform Arabidopsis thaliana ecotypeColumbia and the single, double and triple mutants with constructsharboring the putative melon Mlo orthologs. After selection of thetransformant plants on kanamycin, analyses were performed to identifythe resistant versus susceptible phenotypes upon powdery mildew(Golovinomyces orontii) infection.

Dr. R. Panstruga (MPI, Cologne, Germany) kindly provided Arabidopsisthaliana ecotype Columbia leaves infected with Golovinomyces orontii. Tomaintain a fresh growing Golovinomyces orontii culture, leaves withsporulating Golovinomyces orontii were used every 10-14 days torub-infect fresh Arabidopsis thaliana ecotype Columbia leaves. Inaddition, the mlo single, double and triple mutants and their progeny,complemented with the melon Mlo candidate genes, were inoculated withsporulating Golovinomyces orontii in order to confirm the powdery mildewsusceptibility levels.

Results

To confirm the predicted involvement of the identified CmKIP genes insusceptibility to powdery mildew, full length cDNA sequences of CmKIP1,CmKIP2, CmKIP3 and CmKIP4 were expressed in Arabidopsis using theubiquitous 35S promoter. In addition, also a full length genomicfragment was expressed for CmKIP2 using the 35S promoter.

The 35S::CmKIP1_cDNA, 35S::CmKIP2_cDNA, 35S::CmKIP2_genomic,35S::CmKIP3_cDNA and 35S::CmKIP4_cDNA constructs were transformed toArabidopsis mutant line NACS ID N9707 (mlo2-5 single mutant), N9710(mlo2-5, mlo6-2 double mutant) and N9715 (mlo2-5, mlo6-2, mlo12-1 triplemutant). In addition, to examine epistatic effects of CmKIPoverexpression in Arabidopsis, also WT Columbia plants were transformedwith the same series of constructs.

FIG. 1 and FIG. 2 show the results obtained for the complementationassay for the 35S::KIP1_cDNA construct. The Arabidopsis mutant Atmlo2shows reduced susceptibility compared to WT Columbia (Col-0) (data notshown). The double mutant Atmlo2/6 is even more reduced insusceptibility to Golovinomyces orontii. The triple mutant Atmlo2/6/12is fully resistant to Golovinomyces orontii infection.

Primary transformants of Atmlo2/6/12 with the 35S::CmKIP1_cDNAconstructs retained susceptibility to Golovinomyces orontii infection tothe levels of susceptibility compared to the Atmlo2/6 double mutant.Complementation of the Atmlo2/6 double mutant of Arabidopsis with the35S::CmKIP1 construct resulted in plants with near WT levels ofinfection with Golovinomyces orontii.

A quick and strong development of the fungus resulting in sporulatinghyphae was detected. Complementation of the Atmlo2/6/12 triple mutant ofArabidopsis with the 35S::CmKIP1_cDNA construct resulted in clearvisible powdery mildew infections on inoculated leaves for most of theprimary transformants analyzed. The retaining of susceptibility toGolovinomyces orontii of the Arabidopsis triple mutant previously shownto be full resistant to Golovinomyces orontii indicates that theidentified CmKIP1 gene is able to reestablish the powdery mildewsusceptibility pathway in Arabidopsis. With this result we showed wewere able to identify CmKIP1, a gene functional in the powdery mildewsusceptibility pathway.

FIG. 3 shows the results obtained for the complementation assay for the35S::CmKIP2_cDNA construct. Primary transformants of Atmlo2/6/12 withthe 35S::CmKIP2 cDNA constructs do in a few cases retain minorsusceptibility symptoms to Golovinomyces orontii infection. However,this susceptibility is difficult to detect by eye and not all primarytransformants show this weak susceptibility phenotype. To study in moredetail the development of Golovinomyces orontii, microscopic studieswere performed.

Leaves were washed for 48 hours in 70% EtOH and stained with CBB. Withthis microscopic screening a clear difference was observed between thepowdery mildew resistant AtMlo2/6/12 mutant line and this linecomplemented with 35S::CmKIP2_cDNA.

Of the analyzed complemented lines, several showed clear development ofhyphea in the leaves while all germinating spores on the triple mutantare stopped immediately after penetration in the leaves. On the35S::CmKIP2_cDNA complemented lines the clear development of hyphae wasdetected which indicate that the CmKIP2 gene is able to reestablish thepowdery mildew susceptibility pathway in Arabidopsis.

Comparable results, i.e. complementation in Arabidopsis, was observed,for the CmKIP3 overexpression contruct.

TABLE 2 cDNA and amino acid sequences powdery mildew gene Sequenceidentity Plant type SEQ ID No. CmKIP1 Cucumis melo cDNA 1 aa 2 CmKIP2Cucumis melo cDNA 3 aa 4 CmKIP3 Cucumis melo cDNA 5 aa 6 CmKIP4 Cucumismelo cDNA 7 aa 8 CmKIP6 Cucumis melo cDNA 9 aa 10 CmKIP7 Cucumis melocDNA 11 aa 12 CmKIP9 Cucumis melo cDNA 13 aa 14

1. A full length cDNA transcribed from a nucleotide sequence having thesequence of SEQ ID NO:
 3. 2. A method for obtaining a melon plant thatis resistant to powdery mildew, comprising introducing one or moremutations to a nucleotide sequence having the sequence of SEQ ID NO: 3in a melon plant that is susceptible to powdery mildew, wherein themutation results in a decrease, absence, or loss of function of aprotein having the amino acid sequence of SEQ ID NO: 4 and resistance topowdery mildew.
 3. The method according to claim 2, wherein the one ormore mutations cause the absence of a protein.
 4. The method accordingto claim 2, wherein the one or more mutations cause a non-functioningprotein.
 5. The method according to claim 2, wherein the one or moremutations cause gene silencing.
 6. The method according to claim 2,wherein the one or more mutations are at least three mutations.
 7. Themethod according to claim 2, wherein the resistance is to powdery mildewcaused by Golovinomyces orontii.